Influence of glycosylation on the conformational preferences of folded oligopeptides

Article abstract of DOI:10.1016/S0040-4020(01)00098-9

Synthesis, characterization, and conformational analysis by FT-IR absorption, ¹H NMR and X-ray diffraction techniques are described for a series of side-chain O-glycosylated Thr peptides of different main-chain length rich in the helicogenic Aib residue. The results obtained, compared with those of related peptides containing side-chain protected Thr and Ser residues and host Aib homo-oligomers, also reported in this work, provided new information on the preferred conformation of the naturally occurring antifreeze glycopeptides.

Full text of DOI:10.1016/S0040-4020(01)00098-9

TETRAHEDRON
Pergamon
Tetrahedron 57 (2001) 2433±2443
In¯uence of glycosylation on the conformational preferences of
folded oligopeptides
Marina Gobbo,p Alessia Nicotra, Raniero Rocchi, Marco Crisma and Claudio Toniolo
Biopolymer Research Centre, C. N. R., Department of Organic Chemistry, University of Padova, 35131 Padova, Italy
Received 7 November 2000; revised 15 December 2000; accepted 11 January 2001
1
AbstractÐSynthesis, characterization, and conformational analysis by FT-IR absorption, H NMR and X-ray diffraction techniques are
described for a series of side-chain O-glycosylated Thr peptides of different main-chain length rich in the helicogenic Aib residue. The results
obtained, compared with those of related peptides containing side-chain protected Thr and Ser residues and host Aib homo-oligomers, also
reported in this work, provided new information on the preferred conformation of the naturally occurring antifreeze glycopeptides. q 2001
Elsevier Science Ltd. All rights reserved.
1. Introduction
pattern in water at temperatures higher than 508C or in
2,2,2-tri¯uoroethanol (TFE) was compatible with the
presence of a small amount of helical structure, suggesting
that an ordered conformation (more speci®cally, a 310-
helix4) might form in suf®ciently long segments of glyco-
sylated peptides in a structure supporting solvent. In such a
case the model proposed by Bush and Feeney2 for the anti-
freezing action of AFGPs would still hold, at least partially,
as in the right-handed, threefold 310-helix all sugar side chains
would be located on one side of the rod-like structure.
Antifreeze glycoproteins (AFGPs), present in the serum of
polar and deep-sea ®sh, allow them to survive in seas where
the temperature is sub-zero by depressing the freezing
temperature of the blood without increasing the osmotic
pressure.1 The primary structure of these proteins consist
of repeating glycotripeptide units, (Thr±Ala±Ala)n, with
b-d-galactopyranosyl-(1!3)-a-d-N-acetylgalactosamine
[Gal(b1!3)GalNAca1] disaccharide groups extending
from the Thr side chains. Different fractions of AFGPs
have been isolated, which range from the large AFGP 1
(32 kD) to the smallest AFGP 8, consisting of only four
glyco-tripeptide units (2.7 kD). In order to elucidate their
mechanism of action a number of efforts have been made to
characterize their structure in solution. Bush and Feeney2
proposed an attractive 3D-model for the physiological
action of antifreeze glycoproteins, namely that in water
they form an extended, left-handed threefold helix of
poly(Pro)n II type. In this structure all of the sugar moieties
are on one side of the helix. This disposition allows for
hydrophilic interactions with water molecules to take
place, particularly through the action of the hydroxyl
groups, thus blocking the growth of ice crystals.
In order to prepare well-characterized models of such a
helical structure, we have decided to exploit the known
peculiarity of Ca-tetrasubstituted a-amino acids to stabilize
helical peptide conformations,5,6 by designing repeating
glycotripeptide sequences of the AFGPs in which the Ala
residues are replaced by the prototypical Ca -tetrasubstituted
a-amino acid a-aminoisobutyric acid (Aib). Indeed,
previous work from our as well as from other laboratories5,6
has ®rmly established that, in solvent of low polarity, homo-
oligomers based on Aib (and on other Ca-tetrasubstituted
a-amino acids as well)7 form fully developed 310-helices at
approximately the octamer level, and even from pentamer to
heptamer the amount of 310-helix is already signi®cant. In
the crystal state, however, all terminally blocked Aib homo-
oligomers, beginning from the tripeptide, form either type
III(III0) b-turns,8±10 the basic structural unit of the 310-helix,
or regular 310-helices. Even in hetero-peptides containing
Aib and Ca-trisubstituted protein amino acids, Aib residues
are strong promoters of the 310-helical conformation.5,6,11
Indeed, 310-helices are predominantly adopted by short
(,10 residues) sequences with at least 50% Aib residues
and containing isolated protein amino acids.
CD and NMR studies in water, carried out in our laboratory
on the sequential glycopeptides [Thr(Galb1)±Ala±Ala]n
(nˆ2±7) as models of natural AFGPs, showed no evidence
for the presence of an ordered structure, at least in the
temperature range from 22 to 258C.3 Nevertheless, for the
21-residue glycopeptide [Thr(Galb1)± Ala±Ala]7, the CD
Keywords: conformation; glycopeptides; NMR; peptides; X-ray crystallo-
graphy.
Corresponding author. Tel.: 139-049-827-5741; fax: 139-049-827-
5239; e-mail: gobbo@chor.unipd.it
p
With the aim of determining the in¯uence, if any, of glyco-
sylation on the preferred conformation of folded (helical)
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(01)00098-9

2434
M. Gobbo et al. / Tetrahedron 57 (2001) 2433±2443
the C-terminal H±Aib±OMe (OMe, methoxy)19 or H±
Aib±OtBu (OtBu, tert-butoxy)14 derivative. Z±Aib±OH
(Z, benzyloxycarbonyl) was activated by the symmetrical
anhydride method,15,16 whereas HATU (N-[(dimethyl-
amino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-
methylmethanaminium hexa¯uorophosphate)17 was used as
condensing reagent in couplings involving a chiral Ca-
trisubstituted amino acid, including Z±Thr(GalAc4-
b1)OH.18 In order to avoid premature removal of the Ac
(acetyl) protecting groups from the sugar, in the synthesis of
the Thr glycopeptides (TG) coupling reactions were
stopped after 24 h, even if the amino component was still
detectable. The bis-Thr glycosylated octapeptide 8TG and
the Ser(tBu) (tBu, tert-butyl) pentapeptide 5S were obtained
by fragment condensation of Z±Aib±Aib±OH19 on the Na-
deprotected compounds 6TG and 3S, respectively, in the
presence of HATU. The Z-protecting group was removed
by catalytic hydrogenation.
Table 1. Primary structures of peptides investigated in this work
Symbol
Peptide
3TG
4TG
5TG
6TG
Z±Thr(GalAc4b1) ±Aib±Aib±OtBua
Z±Aib±Thr(GalAc4b1) ±Aib±Aib±OtBu
Z±Aib±Aib±Thr(GalAc4b1) ±Aib±Aib±OtBu
Z±Thr(GalAc4b1) ±Aib±Aib±Thr(GalAc4b1) ±
Aib±Aib±OtBu
8TG
Z±Aib±Aib±Thr(GalAc4b1) ±Aib±Aib±
Thr(GalAc4b1) ±Aib±Aib±OtBu
40TG
3T
4T
5T
3S
Z±Thr(GalAc4b1) ±Aib±Aib±Aib±OMeb
Z±Thr(tBu) ±Aib±Aib±OMec
Z±Aib±Thr(tBu) ±Aib±Aib±OMe
Z±Aib±Aib±Thr(tBu) ±Aib±Aib±OMe
Z±Ser(tBu) ±Aib±Aib±OMe
5S
Z±Aib±Aib±Ser(tBu) ±Aib±Aib±OMe
Z±Aib±Aib±Aib±Aib±OtBu
Z±Aib±Aib±Aib±Aib±Aib±OtBu
Z±Aib±Aib±Aib±Aib±Aib±Aib±OtBu
Z±Aib±Aib±Aib±Aib±Aib±Aib±Aib±Aib±OtBu
4U
5U
6U
8U
a
Z, benzyloxycarbonyl; Ac, acetyl; OtBu, tert-butoxy.
OMe, methoxy.
tBu, tert-butyl.
b
c
Tripeptides were obtained in a pure form by crystallization
and all other compounds were puri®ed by medium-pressure
liquid chromatography. Peptides and glycopeptides were
characterized by melting point determination, optical rota-
tion, thin-layer chromatography (TLC), mass spectra
(MALDI±TOF MS), elemental analysis and 1H NMR.
The synthesis and characterization of the Aib(U) homo-
peptides have already been reported.14
oligopeptides, we have synthesized a series of terminally
protected, O-glycosylated, Aib-rich, Thr peptides (Table
1) and, for comparison, two sets of non-glycosylated
oligopeptides containing side-chain protected Thr or Ser
residues. Their conformational preferences have been
1
investigated by FT-IR absorption, H NMR and, whenever
possible, by X-ray diffraction, and compared with those of
the previously described Aib homo-oligomers.5,6
Preliminary accounts of a limited part of this work have
been reported.12,13
2.2. Crystal structure analysis
The 3d-structure of Z±Aib±Aib±Thr(tBu)± Aib±Aib±
OMe (5T) was determined by X-ray diffraction. Fig. 1
compares the molecular structure of 5T with that recently
reported13 for the related glycosylated tripeptide Z±Thr(Gal-
Ac4b1)± Aib±Aib±OtBu (3TG). Relevant Na-protecting
group, backbone and side-chain torsion angles, as well as
the intra- and intermolecular H-bond parameters, for penta-
peptide 5T are given in Tables 2 and 3, respectively.
2. Results and discussion
2.1. Synthesis and characterization
Peptides and glycopeptides were synthesized in solution,
preferentially by the step-by-step approach, starting from
Bond lengths and bond angles are in general agreement with
Figure 1. X-ray diffraction structures of Z±Aib±Aib±Thr(tBu)±Aib±Aib±OMe (5T) (left) and Z±Thr(GalAc4b1)±Aib±Aib±OtBu (3TG) (Ref. 13) (right).
The intramolecular H-bonds are represented by dashed lines.

M. Gobbo et al. / Tetrahedron 57 (2001) 2433±2443
2435
Table 2. Selected Na-protecting group, backbone and side-chain torsion
angles (deg) [the torsion angles for rotation about bonds of the peptide
backbone (f, c, v) and side-chains (x) are described in Ref. 44. For the
torsion angles for rotation about bonds of the Z-protecting group (u) see
Ref. 20] for Z±Aib±Aib±Thr(tBu) ±Aib±Aib±OMe (5T)
The molecules of the helical pentapeptide are held together
in a head-to-tail fashion along the a-direction, in rows
stabilized by (urethane) N1±H1´´´O4vC4 (peptide) inter-
molecular H-bonds. The additional potential donor N2±H2
group does not seem to be involved in the intermolecular
H-bonding scheme.
Torsion angle
Peptide 5T
Torsion angle
Peptide 5T
2
u
u
298.7(3)
2167.5(3)
2170.2(3)
256.7(4)
229.4(4)
2175.5(3)
254.7(4)
227.7(4)
2177.9 (2)
266.7(3)
214.0(3)
2172.9(2)
f4
c4
v4
f5
256.4(3)
225.7(4)
178.2(3)
47.0(4)
1
Initial information on the in¯uence of glycosylation on the
preferred conformation of peptides rich in the strong turn/
helical forming Aib residue5,6,11 is provided by a comparison
of the X-ray diffraction structures of the non-glycosylated
pentapeptide 5T and its glycosylated, C-terminal tripeptide
short sequence (3TG)13 (Fig. 1). It is clear that the presence
of an internal Thr(tBu) residue does not signi®cantly perturb
the crystal-state helical structure of 5T, despite the bulki-
ness and H-bonding acceptor properties of its ether side-
chain moiety. By contrast, owing to the occurrence of
side-chain to main-chain, carbohydrate-to-peptide intra-
molecular H-bonds, the peptide backbone of the tripeptide
with an N-terminal Thr(GalAc4b1) residue is forced to
adopt a more extended conformation than the expected
regularly folded structure. It remains to be seen whether
this dramatic conformational in¯uence will also be related
to the length of the peptide (i.e. to the intrinsic stability of
the turn/helix) in general and to the position of the glyco-
sylated residue in the peptide main chain in particular.
v0
f1
c1
v1
f2
c2
v2
f3
c3
v3
a
c5
v5
47.0(3)
b
2176.3(3)
260.8(3)
61.1(3)
x1,1
c
3
d
x1,2
3
x2
139.0(2)
3
a
N5-C5A-C5-OT torsion angle.
C5A-C5-OT-CT torsion angle.
N3-C3A-C3B-C3G torsion angle.
N3-C3A-C3B-O3G torsion angle.
b
c
d
previously reported values for the geometry of the benzyl-
oxycarbonylamino moiety,20 the methyl ester group,21 the
Aib residue,22,23 and the peptide unit.24,25 The N3±C3A±C3
bond angle of the Thr(tBu) residue is signi®cantly expanded
relative to the regular tetrahedral value, 114.2(2)8, presum-
ably to help accomodate the bulky ether side chain.
2.3. Solution conformational analysis
The backbone of the pentapeptide is folded in a right-
handed 310-helical structure.4 Peptide groups N3±H3 to
N5±H5 and O0vC0 to O2vC2 participate in three con-
secutive 1Ã4 (type III b-turn8±10) CvO´´´H±N intra-
molecular H-bonds, appropriate for a 310-helix. The range
The conformational preferences of the four series of termi-
nally and side-chain protected Aib-rich peptides containing
O-glycosylated Thr (3-6TG, 8TG and 40TG), Thr (3-5T)
and Ser (3S and 5S), respectively, were investigated in a
solvent of low polarity (CDCl3) at different concentrations
(in the range 10±0.1 mM) by using FT-IR absorption and 1H
NMR techniques. Figs. 2 and 3 show the most relevant
FT-IR absorption spectra in the conformationally informa-
tive 3500±3200 cm21 region (amide N±H stretching or
amide A) region. Using Mizushima's dilution method31
we have been able to show that at 1 mM concentration
self-association via N±H´´´OvC intermolecular H-bonding
is negligible for all oligopeptides (spectra not shown). As a
consequence, for all peptides investigated, the H-bonding
observed at 1 mM concentration should be interpreted as
arising from intramolecular interactions only.
Ê
of observed N´´´O distances, 3.008(3)± 3.187(3) A, and that
of N±H´´´O angles, 164.1±174.48, are those usually seen for
such intramolecular H-bonds.26±28 Also the C-terminal Aib
residue adopts a conformation in the helical region, but it
has a handedness opposite to that shown by the preceding
residues, a common observation for Aib-rich 310-helical
peptides.6
In the pentapeptide no signi®cant deviation of the v torsion
angles (.108) from the ideal value of the trans planar
urethane, peptide, and ester units (1808) is observed. The
trans arrangement of the u1 torsion angle of the benzyl-
oxycarbonylamino moiety is that usually reported for
Z-amino acids and peptides.20 Also the value of u2,
298.7(3)8, is found in one of the expected regions (^908,
1808).20 The methyl ester conformation with respect to
preceding Ca±N bond is intermediate between the anticlinal
and antiperiplanar conformations.29 The set of x1 torsion
angles found for the Thr side chain (g2 g1, with the Og atom
in the g1 position) is that preferred by Thr peptides.30
In the TG series, Fig. 2(I), the curves are complex and
characterized by several absorptions.32 We assign: (i) the
band (shoulder) at about 3430 cm21, present only in
the spectra of the peptides with an N-terminal Aib
residue, to free, solvated NH groups; (ii) the band(s) in
the 3410±3390 cm21 region, relatively stronger in the
peptides with an N-terminal Thr(GalAc4b1) residue, to
weakly H-bonded NH groups typical of the fully-extended
Table 3. Intra- and intermolecular H-bond parameters for Z±Aib±Aib±Thr(tBu)±Aib±Aib-OMe (5T)
Ê
Distance (A) D¼A
Ê
Distance (A) H¼A
Donor D±H
Acceptor A
Symmetry operation
Angle (deg) D±H¼A
N3±H3
N4±H4
N5±H5
N1±H1
O0
O1
O2
O4
x, y, z
x, y, z
x, y, z
x11, y, z
3.187(3)
3.008(3)
3.102(3)
2.826(3)
2.33
2.17
2.26
1.99
174.4
164.1
165.6
164.0

2436
M. Gobbo et al. / Tetrahedron 57 (2001) 2433±2443
Figure 2. FT-IR absorption spectra (N±H stretching region) of the glycosylated peptides 3TG, 4TG, 5TG, 6TG and 8TG (I) and the Aib homo-peptides 4U,
5U, 6U and 8U (II) in CDCl3 solution. Peptide concentration: 1 mM.
(C5) conformation9,33 and, probably, of side-chain to main-
chain interactions as well;34,35 (iii) the pronounced shoulder
at 3360±3330 cm21, shown by the 4TG±8TG peptides, to
strongly H-bonded NH groups typical of folded conforma-
tions. Conversely, the spectra of the Aib homo-oligomers,
Fig. 2(II), are much simpler, exhibiting only the two bands
at about 3430 and 3350 cm21, indicative of an increasing
amount of folded forms with main-chain elongation.36,37
An inspection of the spectra of peptides with the same main-
chain length (either tetra- or pentapeptides), illustrated in
Fig. 3, allow a more stringent comparison of the conforma-
tional propensities of the various series to be made. Clearly,
the observed decreasing rank order of the intensity of the
<3350 cm21 band relative to the bands of free/weakly
H-bonded NH groups at 3430±3390 cm21 (Aib.Ser(t-
Bu).Thr(tBu)qThr(GalAc4b1)) strongly supports the
Figure 3. FT-IR absorption spectra (N±H stretching region) of the tetrapeptides 4TG, 40TG, 4T and 4U (I) and the pentapeptides 5TG, 5T, 5S and 5U (II) in
CDCl3 solution. Peptide concentration: 1 mM.

M. Gobbo et al. / Tetrahedron 57 (2001) 2433±2443
2437
Figure 4. Plot of NH chemical shifts (d) in the 1H NMR spectra of the pentapeptides 5TG, 5T, 5S and 5U as a function of increasing percentages of DMSO-d6
added to the CDCl3 solution (v/v). Peptide concentration: 1 mM.
view that the Thr(GalAc4b1) residues effectively interfere
with the propensity of the Aib-rich segments to fold into
turn/helical structures. This phenomenon is slightly more
signi®cant when the glycosylated Thr residue is positioned
at the N-terminus of the peptide chain (compare the curves
of the two isomeric tetrapeptides 4TG and 40TG). In
conclusion, a combination of side-chain bulkiness and
appropriate location of H-bonding acceptor groups seems
to drastically reduce the extent of turn/helix formation
even in a structure supporting solvent and in Aib-based
peptides.
tion of inaccessible (or intramolecular H-bonded) NH
groups by H NMR was performed using solvent depen-
1
dence of NH chemical shifts, by adding increasing
amounts of the strong H-bonding acceptor solvent dimethyl-
sulphoxide (DMSO) to the CDCl3 solution (peptide concen-
tration 1 mM).38,39 As typical examples, Figs. 4 and 5
illustrate the behaviour of the NH resonances of the
penta-, hexa-, and octapeptides. Assignment of the confor-
mationally critical N(1)H and N(2)H protons was achieved
by chemical shift analysis (the position of the N-terminal
urethane proton is always largely up®eld with respect to the
peptide protons)32 and ROESY experiments.
To obtain more detailed information on the conformational
preferences of the four peptide series in CDCl3 solution, we
carried out a 400 MHz 1H NMR investigation. The delinea-
In all of the peptides shown in Figs. 4 and 5, except 6TG,
two classes of NH protons were observed. Class (a) [N(1)H

2438
M. Gobbo et al. / Tetrahedron 57 (2001) 2433±2443
Figure 5. Plot of NH chemical shifts (d) in the 1H NMR spectra of the hexapeptides 6TG and 6U, and the octapeptides 8TG and 8U as a function of increasing
percentages of DMSO-d6 added to the CDCl3 solution (v/v). Peptide concentration: 1 mM.
and N(2)H protons] includes protons whose chemical shifts
are sensitive to the addition of DMSO. Interestingly, the
sensitivity of the N(1)H proton is much higher than that of
the N(2)H proton. Class (b) [all other NH protons] includes
those displaying a behaviour characteristic of shielded
protons (relative insensitivity of chemical shifts to solvent
composition). However, in hexapeptide 6TG, with a glyco-
sylated Thr residue at the N-terminus, even the chemical
shift of the N(1)H proton does not change signi®cantly
upon addition of DMSO. Between 0 and 10% DMSO in
CDCl3 (v/v) the decreasing rank orders of D ppm values
for N(1)H protons of peptides of the same main-chain length
are as follows: 5U.5S.5T.5TG; 6Uq.6TG;
8Uq8TG. The same trend is seen for the D ppm values
of the N(2)H protons.
In summary, these 1H NMR results allow us to conclude that
in the structure-supporting solvent CDCl3 the N(3)H protons
and the following NH protons in the main-chain of the four
peptide series investigated are almost inaccessible to the
perturbing agent and are therefore most probably intra-
molecularly H-bonded. From the analysis of the behaviour
of the N(1)H and N(2)H protons, however, it seem safe to
conclude that the stability of the regular 310-helical structure
typical of the (Aib)n homo-oligomers (where only the two
N-terminal NH protons do not participate in the intra-
molecular H-bonding scheme) is only slightly reduced by
the presence of one Ser(tBu) or Thr(tBu) residue, whereas
one (or two) Thr(GalAc4b1) residue(s) is (are) highly detri-
mental, particular when located at the N-terminus of the
peptide main-chain. It is worth noting that these conclusions

M. Gobbo et al. / Tetrahedron 57 (2001) 2433±2443
2439
of the 1H NMR study agree well with those extracted from
the FT-IR absorption investigation discussed above.
silica gel 60 (0.040±0.063 mm) using a BuÈchi 688 chroma-
tographic pump connected with a BuÈchi UV/Vis ®lter
photometer detector and a Knauer recorder (see text for
elution conditions). Melting points were taken on a BuÈchi
model 150 apparatus in open capillary, at 208C, and are not
corrected. Optical rotations were recorded on a Perkin±
Elmer 241 polarimeter. NMR spectra were obtained either
on a Bruker AM-400 or on a Bruker AC-250F spectrometer
at 298 K in CDCl3. Chemical shifts are given in ppm and
referred to tetramethylsilane as the internal standard. For the
hexa- and octaglycopeptides the assignments of the 1H
NMR resonances were based on 2D proton±proton shift
correlation spectra. MALDI±TOF MS was carried out on
a Bruker Re¯ex TOF instrument, operating in the linear
mode at nominal accelerating potential of 120 kV (matrix:
a-cyano-4-hydroxycinnamic acid).
3. Conclusions
Clearly, the repetition of a triplet, ±Thr(O±glycosyl)± Ala±
Ala±, in the sequential peptides with antifreeze activity
from Antartic ®sh1,2 implies a ternary helix as a reasonable
conformation. Of the two most extensively described
ternary helices in peptides and proteins, the extended
poly(Pro)n II helix and the folded 310-helix,4 recent confor-
mational work40 tends to favour the former as the character-
istic structural motif of the antifreeze peptides. However,
the contribution of the latter type of helix could not be
completely discarded.3
4.1.1. N-(Benzyloxycarbonyl)-O-(2,3,4,6-tetra-O-acetyl-
a-d-galactopyranosyl)-l-threonyl-a-aminoisobutyryl-a-
aminoisobutyric acid tert-butyl ester [Z±Thr(Ga-
lAc4b1)± Aib±Aib±OtBu] (3TG). To an ice-cooled solu-
tion of Z±Thr(GalAc4b1)±OH18 (2.95 g, 5 mmol) and
HATU (1.96 g, 5 mmol) in CH2Cl2/DMF (4:1, 5 ml),
2.4 ml of diisopropylethylamine (DIEA), H±Aib±Aib±
OtBu14 (0.81 g, 3.3 mmol) and HOAt (0.46 g, 3.3 mmol)
were consecutively added and the reaction mixture was
stirred at room temperature. After 24 h the solvent was
evaporated in vacuo, the residue taken up with EtOAc and
washed with 0.5 M citric acid (2£25 ml), saturated NaCl
(1£25 ml), 5% NaHCO3 (3£25 ml), and H2O (2£25 ml).
The organic layer was dried over Na2SO4 and evaporated
to dryness. The title compound was obtained as a white solid
after puri®cation by MPLC (1: 1 EtOAc/petroleum ether).
Yield 1.5 g (57%); Rf (E6) 0.71; Rf (E8) 0.80; mp 160±
1618C; (Found: C, 56.29; H, 6.78; N, 5.15%. C38H55N3O16
requires C, 56.36; H, 6.85; N, 5.19%); [a]Dˆ19.4 (c ˆ1.0,
MeOH); dH (400 MHz, CDCl3): 7.34 (5H, m, Ph CH), 7.04
(1H, s, Aib NH), 6.86 (1H, s, Aib NH), 5.88 (1H, d,
Jˆ6.0 Hz, Thr NH), 5.41 (1H, d, Jˆ3.0 Hz, Gal H4),
5.18±5.05 (2H, m, Gal H2 and H3), 5.08 (2H, s, CH2Ph),
4.72 (1H, d, Jˆ7.9 Hz, Gal H1), 4.43 (1H, m, Thr CaH), 4.29
(1H, m, Gal H6), 4.10 (3H, m, Thr CbH, Gal H6', Gal H5),
2.13, 2.05, 2.00, 1.99 (12H, 4s, 4 GalAc CH3±CO), 1.59±
1.46 (12H, 4s, 4 Aib CbH3), 1.44 (9H, s, OtBu CH3), 1.13
(3H, d, Jˆ6.4 Hz, Thr CgH3); MALDI±TOFMS: [M1Na]1,
found 832. C38H55N3O16 requires 809.8.
With the aim of shedding light on the contribution, if any, of
the 310-helix to the 3d-structural ensemble of antifreeze
peptides, in this work we examined the solution preferred
conformations of peptides containing one (or two)
Thr(GalAc4b1) residue(s) and characterized by a high
amount of Aib residues. The incorporation of this strongly
helicogenic amino acid5,6 was intended to drastically bias
the peptide conformation towards the formation of a
310-helical structure. In our model compounds the acetoxy
groups of the peracetylated, O-glycosylated Thr side chain
are expected to mimic the acetamido group of the GalNAc
moiety of the disaccharide unit present in the antifreeze
peptides.1,2
Taken together, the results described here, combined with
our crystallographic data illustrated in Ref. 13, are strongly
in favour of the thesis that the presence of a glycosylated
Thr residue tends to discourage the formation of a 310-heli-
cal structure even in an Aib-rich peptide, particularly when
the glycosylated residue is located at the N-terminus of the
main chain, and that the major factor operative in the helix
destabilization is the competitive role of side-chain to main-
chain H-bonding. In our view this conclusion implies that
the occurrence of the 310-helix to a signi®cant extent in the
conformational equilibrium mixture of the antifreeze
peptides, where the much less helicogenic Ala residues
are found in lieu of the Aib residues, should be regarded
as a remote possibility.
4.1.2. N-(Benzyloxycarbonyl)-a-aminoisobutyryl-O-(2,3,
4,6-tetra-O-acetyl-a-d-galactopyranosyl)-l-threonyl-a-
aminoisobutyryl-a-aminoisobutyric acid tert-butyl ester
[Z±Aib±Thr(GalAc4b1)± Aib±Aib±OtBu] (4TG). To a
chilled solution of Na-deprotected tripeptide 3TG (1.1 g,
1.6 mmol) in 10 ml CH2Cl2, (Z±Aib)2O15,16 (0.90 g,
1.9 mmol) and DIEA (0.34 ml) were added and the reaction
mixture was stirred at room temperature for 24 h. The
solvent was removed in vacuo and the oily residue taken
up with EtOAc (30 ml) and worked up as described for
compound 3TG. Puri®cation by MPLC (75: 25 EtOAc/
petroleum ether) yielded 110 mg (53%) of the title
compound as a white solid. Rf (E6) 0.67; Rf (E8) 0.60; mp
132±1338C; (Found: C, 56.35; H, 6.95; N, 6.25%.
C42H62N4O17 requires C, 56.37; H, 6.98; N, 6.26%);
[a]Dˆ22.9 (cˆ1.0, MeOH); dH (400 MHz, CDCl3): 7.34
4. Experimental
4.1. General procedures and materials
HATU and HOAt (1-hydroxy-7-azabenzotriazole) were
purchased from PerSeptive Biosystems Gmbh. All other
chemicals were of the best grade commercially available.
TLC was performed on Merck silica gel 60 F254 precoated
plates using the following solvent systems: E6: CHCl3/
MeOH (methanol)/acetic acid (90:8:2); E8: EtOAc (ethyl
acetate).
Amino acid derivatives and peptides were detected by one
or more of the following procedures: ninhydrin, 10% sul-
furic acid in ethanol (1008C) or UV light. Medium-pressure
liquid chromatography (MPLC) was performed on Merck

2440
M. Gobbo et al. / Tetrahedron 57 (2001) 2433±2443
(5H, m, Ph CH), 7.05 (2H, m, Thr NH and Aib NH), 7.00
(1H, s, Aib NH), 5.41 (1H, d, Jˆ3.0 Hz, Gal H4), 5.25 (1H,
s, Aib NH), 5.16 (1H, dd, Jˆ3.0, 10.5 Hz, Gal H3), 5.09 (2H,
s, CH2Ph), 5.04 (1H, dd, Jˆ7.8, 10.5 Hz, Gal H2), 4.66 (1H,
MPLC (75:25 EtOAc/petroleum ether),as a white solid.
Yield 110 mg (84%); Rf (E6) 0.57; Rf (E8) 0.57; mp 119±
1218C; (Found: C, 54.26; H, 6.73; N, 6.17%. C64H94N6O29
requires C, 54.46; H, 6.71; N, 5.95%); [a]Dˆ110.9 (cˆ1.0,
MeOH); dH (400 MHz, CDCl3): 7.34 (5H, m, Ph CH),
7.27 (1H, s, Aib NH), 7.15 (1H, d, Jˆ7.0 Hz, ThrA
NH), 7.12 (1H, s, Aib NH), 6.99 (2H, broad s, 2 Aib
NH), 6.38 (1H, d, ThrB NH), 5.43 (1H, d, Jˆ2.9 Hz,
GalA H4), 5.39 (1H, d, Jˆ3.2 Hz, GalB H4), 5.20±5.00
(6H, m, GalA H2, GalB H2, GalA H3, GalB H3, CH2Ph),
4.73 (1H, d, Jˆ7.8 Hz, GalA H1), 4.70 (1H, d, Jˆ7.6 Hz,
GalB H1), 4.57 (2H, m, ThrA CaH, GalA H6), 4.35 (1H, m,
ThrB CaH), 4.24 (2H, m, ThrA CbH, GalA H5), 4.10±4.03
d, Jˆ7.8 Hz, Gal H1), 4.45 (1H, m, Thr CaH), 4.28 (1H, m,
b
0
Gal H6), 4.20 (1H, m, Thr C H), 4.09 (1H, m, Gal H6 ), 4.03
(1H, m, Gal H5), 2.09, 2.05, 2.03, 2.00 (12H, 4s, 4 GalAc
CH3±CO), 1.54, 1.51, 1.50, 1.49, 1.47, 1.46 (18H, 6s, 6 Aib
CbH3), 1.44 (9H, s, OtBu CH3), 1.10 (3H, d, Jˆ6.0 Hz, Thr
CgH3); MALDI±TOFMS: [M1Na]1, 918. C42H62N4O17
requires 894.9.
4.1.3. N-(Benzyloxycarbonyl)-a-aminoisobutyryl-a-amino-
isobutyryl-O-(2,3,4,6-tetra-O-acetyl-a-d-galactopyrano-
syl)-l-threonyl-a-aminoisobutyryl-a-aminoisobutyric
acid tert-butyl ester [Z±Aib±Aib±Thr(GalAc4b1)± Aib±
Aib±OtBu] (5TG). (a) Stepwise procedure.The Na-depro-
tected tetrapeptide 4TG (0.30 g, 0.39 mmol) was acylated
with (Z±Aib)2O and the crude product was worked up as
described above for 4TG. The title compound was obtained
as a white solid after MPLC puri®cation (7:3 EtOAc/
petroleum ether). Yield 0.146 g (38%); Rf (E6) 0.66; Rf
(E8) 0.60; mp 95±978C; (Found: C, 56.24; H, 7.15; N,
6.99%. C46H69N5O18 requires C, 56.37; H, 7.10; N,
7.15%); [a]Dˆ18.5 (c ˆ0.9, MeOH); dH (400 MHz,
CDCl3): 7.35 (5H, m, Ph CH), 7.09 (1H, s, Aib NH), 6.98
(1H, s, Aib NH), 6.95 (1H, d, Thr NH), 6.89 (1H, s, Aib
NH), 5.38 (1H, d, Jˆ3.2 Hz, Gal H4), 5.31 (1H, s, Aib NH),
5.13±5.08 (3H, m, Gal H2 and CH2Ph), 5.02 (1H, dd, Jˆ3.3,
10.5 Hz, Gal H3), 4.65 (1H, d, Jˆ7.8 Hz, Gal H1), 4.43 (1H,
m, Thr CaH), 4.28 (1H, m, Thr CbH), 4.20 (1H, dd, Gal H6),
B
b
B
B
A
0
0
(4H, m, Thr C H, Gal H6, Gal H6 , Gal H6 ), 3.91 (1H,
m, GalB H5), 2.14, 2.13, 2.11, 2.04, 2.03, 2.00, 1.98, 1.90
(24H, 8s, 8 GalAc CH3±CO), 1.55±1.41 (24H, m, 8 Aib
CbH3), 1.43 (9H, s, OtBu CH3), 1.20 (3H, d, Jˆ6.8 Hz,
ThrA CgH3); 1.11 (3H, d, Jˆ5.7 Hz, ThrB CgH3);
MALDI±TOFMS: [M1Na]1 found 1435. C64H94N6O29
requires 1411.5.
(b) Fragment condensation. Compound 3TG (0.15 g,
0.19 mmol) was dissolved in 5 ml of ice-cooled 90% aq
TFA and stirred at room temperature for 60 min. The
solvent was evaporated in vacuo and the oily residue was
taken up with EtOAc (20 ml), washed several times with
water, dried (Na2SO4) and evaporated to dryness. The
resulting Z±Thr(GalAc4b1)± Aib±Aib±OH (0.13 g,
0.40 mmol) was coupled with Na-deprotected 3TG as
described above for the synthesis of 5TG by fragment
condensation (b). The title compound was obtained, after
puri®cation by MPLC, as a white solid, in 62% yield.
0
4.08 (1H, dd, Gal H6 ), 4.02 (1H, m, Gal H5), 2.09, 2.04,
2.03, 1.98 (12H, 4s, 4 GalAc CH3±CO), 1.58±1.45 (24H, m,
8 Aib CbH3), 1.43 (9H, s, OtBu CH3), 1.14 (3H, d,
Jˆ6.2 Hz, Thr CgH3); MALDI±TOFMS: [M1Na]1 found
1003. C46H69N5O18 requires 980.0.
4.1.5. N-(Benzyloxycarbonyl)-a-aminoisobutyryl-a-amino-
isobutyryl-O-(2,3,4,6-tetra-O-acetyl-a-d-galactopyrano-
syl)-l-threonyl-a-aminoisobutyryl-a-aminoisobutyryl-
O-(2,3,4,6-tetra-O-acetyl-a-d-galactopyranosyl)-l-thre-
onyl-a-aminoisobutyryl-a-aminoisobutyric acid tert-
butyl ester [Z±Aib±Aib±Thr(GalAc4b 1)± Aib±Aib±
Thr(GalAc4b1)± Aib±Aib±OtBu] (8TG). The title
compound was prepared from Z±Aib±Aib±OH (87 mg,
0.27 mmol) and the N a-deprotected hexapeptide 6TG
(100 mg, 0.08 mmol) as described above for the synthesis
of 5TG by fragment condensation (b), and obtained, after
puri®cation by MPLC (9:1 EtOAc/petroleum ether), as a
white solid. Yield 32 mg (25%); Rf (E6) 0.61; Rf (E8)
0.22; mp 125±1288C; (Found: C, 54.42; H, 7.00; N,
7.05%. C72H108N8O31 requires C, 54.68; H, 6.88; N,
7.08%); [a]Dˆ19.2 (c ˆ1.0, MeOH); dH (400 MHz,
CDCl3): 7.35 (5H, m, Ph CH), 7.23 (1H, s, Aib NH), 7.16
(2H, m, Aib NH, ThrA NH), 7.08 (2H, m, Aib NH, ThrB
NH), 6.90 (2H, broad s, 2 Aib NH), 5.97 (1H, s, Aib NH),
5.40 (2H, m, GalA H4, GalB H4), 5.15±5.08 (4H, m, GalA H2,
GalB H2, CH2Ph), 5.03 (2H, m, GalA H3, GalB H3), 4.79 (1H,
d, Jˆ7.9 Hz, GalA H1), 4.65 (1H, broad d, ThrA CaH, GalB
H1), 4.52 (1H, m, GalA H5), 4.36 (1H, m, ThrB CaH,), 4.17
(2H, m, ThrA CbH, ThrB CbH), 4.09 (2H, m, GalB H5, GalA
(b) Fragment condensation. Z±Aib±Aib±OtBu14 (0.2 g,
0.54 mmol) was dissolved in 5 ml of ice-cooled 90% aq
TFA (tri¯uoroacetic acid) and stirred at room temperature
for 30 min. The solvent was evaporated in vacuo and the
residue was triturated several times with diethyl ether
(Et2O) and dried. The resulting Z±Aib±Aib±OH19 (0.16 g,
0.40 mmol) was added to a chilled solution of Na-depro-
tected tripeptide 3TG (0.18 g, 0.27 mmol), HATU (0.16 g,
0.42 mmol) and DIEA (0.14 ml) in 5 ml of CH2Cl2/DMF
(4:1). The reaction mixture was stirred at room temperature,
by keeping the pH at 7±8 by addition of DIEA. After 24 h
the solvent was removed under reduced pressure and the
residue worked up as described for compound 3TG. The
title compound was obtained, after MPLC puri®cation, as
a white solid, in 26% yield.
4.1.4. N-(Benzyloxycarbonyl)-O-(2,3,4,6-tetra-O-acetyl-
a-d-galactopyranosyl)-l-threonyl-a-aminoisobutyryl-a-
aminoisobutyryl-O-(2,3,4,6-tetra-O-acetyl-a-d-galacto-
pyranosyl)-l-threonyl-a-aminoisobutyryl-a-aminoiso-
butyric acid tert-butyl ester [Z±Thr(GalAc4b1)± Aib±
Aib±Thr(GalAc4b1)± Aib±Aib±OtBu] (6TG). (a) Step-
wise procedure. The Na-deprotected pentapeptide 5TG
(80 mg, 0.10 mmol) was reacted with Z±Thr(GalAc4b1)±
OH (90 mg, 0.16 mmol) as described above for 3TG, and
the resulting hexapeptide was obtained, after puri®cation by
A
B
0
H6), 3.96 (1H, m, Gal H6 ), 3.90 (1H, m, Gal H6), 2.13±
1.99 (24H, 8s, 8 GalAc CH3±CO), 1.54±1.36 (36H, m, 12
Aib CbH3), 1.43 (9H, s, OtBu CH3), 1.18 (3H, d, Jˆ6.8 Hz,
ThrA CgH3); 1.10 (3H, d, Jˆ6.8 Hz, ThrB CgH3); MALDI±
TOFMS: [M1Na]1 found 1605. C72H108N8O31 requires
1581.7.

M. Gobbo et al. / Tetrahedron 57 (2001) 2433±2443
2441
4.1.6. N-(Benzyloxycarbonyl)-O-(2,3,4,6-tetra-O-acetyl-
a-d-galactopyranosyl)-l-threonyl-a-aminoisobutyryl-a-
aminoisobutyryl-a-aminoisobutyric acid methyl ester
[Z±Thr(GalAc4b1)± Aib±Aib±Aib±OMe] (40TG). Z±
Aib±Aib±Aib±OMe19 (150 mg, 0.35 mmol) was Na-depro-
tected, dissolved in 2 ml of CH2Cl2 and added to a chilled
solution of Z±Thr(GalAc4b1)± OH (306 mg, 0.52 mmol),
HATU (199 mg, 0.52 mmol), HOAt (42 mg, 0.31 mmol)
and DIEA (0.22 ml) in 3 ml of CH2Cl2/DMF (2:1). After
48 h the solvent was evaporated in vacuo and the oily resi-
due worked up as described above for 3TG. The title
compound was obtained as a white solid after puri®cation
by MPLC (8:2 EtOAc/petroleum ether). Yield 214 mg
(75%); Rf (E6) 0.93; Rf (E8) 0.62; mp 96±988C; (Found:
C, 54.79; H, 6.90; N, 6.42%. C39H56N4O17 requires C, 54.92;
H, 6.62; N, 6.57%]; [a]Dˆ13.8 (c ˆ1.0, MeOH); dH
(400 MHz, CDCl3): 7.35 (5H, m, Ph CH), 7.20, 6.96, 6.80
(3H, 3s, 3 Aib NH), 5.73 (1H, d, Jˆ5.7 Hz, Thr NH), 5.44
(1H, d, Jˆ3.0 Hz, Gal H4), 5.17 (1H, m, Gal H2), 5.09 (2H,
s, CH2Ph), 5.07 (1H, dd, Jˆ15.0 Hz, Jˆ16.0 Hz, Gal H3),
4.72 (1H, d, Jˆ8.0 Hz, Gal H1), 4.57 (1H, m, Gal H6), 4.41
(1H, m, Thr CaH), 4.07 (2H, m, Thr CbH, Gal H5), 3.94 (1H,
isobutyryl-O-(tert-butyl)-l-threonyl-a-aminoisobutyryl-
a-aminoisobutyric acid methyl ester [Z±Aib±Aib±
Thr(tBu)± Aib±Aib±OMe] (5T). The N a-deprotected
tetrapeptide 4T (0.30 g, 0.70 mmol) was reacted with (Z±
Aib)2O according to the procedure described above for
4TG, and the resulting pentapeptide was obtained as a
white solid, after puri®cation by MPLC (98: 2 CH2Cl2/
MeOH). Yield 0.27 g (60%); Rf (E6) 0.82; Rf (E8) 0.77;
mp 199±2008C; (Found: C, 59.76; H, 8.27; N, 10.35%.
C33H53N5O9 requires C, 59.71; H, 8.05; N, 10.55%);
[a]Dˆ121.3 (cˆ1.0, MeOH); dH (400 MHz, CDCl3):
7.40 (1H, s, Aib NH), 7.36 (5H, m, Ph CH), 7.08 (2H, m,
Aib NH, Thr NH), 6.93 (1H, broad s, Aib NH), 5.18 (1H, s,
Aib NH), 5.12 (2H, q, CH2Ph), 4.33 (1H, m, Thr CbH), 4.01
(1H, m, Thr CaH), 3.69 (3H, s, OMe CH3), 1.56±1.46 (24H,
m, 8 Aib CbH3), 1.15 (9H, s, tBu CH3), 1.12 (3H, d,
Jˆ6.5 Hz, Thr CgH3); MALDI±TOFMS: [M1Na]1 found
686. C33H53N5O9 requires 663.8.
4.1.10. N-(Benzyloxycarbonyl)-O-(tert-butyl)-l-seryl-a-
aminoisobutyryl-a-aminoisobutyric acid methyl ester
[Z±Ser(tBu)± Aib±Aib±OMe] (3S). This compound was
prepared from Z±Ser(tBu)± OH44 (0.80 g, 2.72 mmol) and
H±Aib±Aib±OMe (0.55 g, 2.72 mmol) as described above
for 3TG, and puri®ed by MPLC (99.5:0.5 CH2Cl2/MeOH).
Yield 1.25 g (95%); colourless oil; Rf (E6) 0.96; Rf (E8)
0.57; (Found: C, 60.05; H, 7.81; N, 8.70%. C24H37N3O7
requires C, 60.11; H, 7.78; N, 8.76%); [a]Dˆ18.4
(cˆ0.6, MeOH); dH (400 MHz, CDCl3): 7.36 (5H, m, Ph
CH), 7.12, 6.86 (2H, 2s, 2 Aib NH), 5.66 (1H, broad s, Ser
NH), 5.12 (2H, q, CH2Ph), 4.13 (1H, m, Ser CaH), 3.79 (1H,
m, Ser CbH), 3.70 (3H, s, OMe CH3), 3.41 (1H, m, Ser
CbH0), 1.58, 1.52, 1.50, 1.47 (12H, 4s, 4 Aib CbH3), 1.20
(9H, s, tBu CH3).
0
dd, Gal H6 ), 3.69 (3H, s, OMe CH3), 2.13, 2.05, 2.00, 1.98
(12H, 4s, 4 GalAc CH3±CO), 1.51±1.44 (18H, m, 6 Aib
CbH3), 1.13 (3H, d, Jˆ6.3 Hz, Thr CgH3); MALDI±
TOFMS: [M1Na]1 found 875. C39H56N4O17 requires 852.9.
4.1.7. N-(Benzyloxycarbonyl)-O-(tert-butyl)-l-threonyl-
a-aminoisobutyryl-a-aminoisobutyric acid methyl
ester [Z±Thr(tBu)± Aib±Aib±OMe] (3T). The title
compound was prepared from Z±Thr(tBu)± OH41 (0.82 g,
2.67 mmol) and H±Aib±Aib±OMe19 (0.54 g, 2.67 mmol)
as described above for 3TG and puri®ed by MPLC (99.5:
0.5 CH2Cl2/MeOH). Yield 0.93 g (64%); colourless oil; Rf
(E6) 0.97; Rf (E8) 0.93; (Found: C, 60.95; H, 8.10; N,
8.27%. C25H39N3O7 requires C, 60.83; H, 7.96; N, 8.51%);
[a]Dˆ125.8 (c ˆ1.1, MeOH); dH (250 MHz, CDCl3): 7.35
(5H, m, Ph CH), 7.30 (2H, s, 2 Aib NH), 5.90 (1H, d,
Jˆ4.8 Hz, Thr NH), 5.12 (2H, q, CH2Ph), 4.15 (2H, m,
Thr CaH, Thr CbH), 3.70 (3H, s, OMe CH3), 1.54,
1.52.1.50, 1.49 (12H, 4s, 4 Aib CbH3), 1.30 (9H, s, tBu
CH3), 1.07 (3H, d, Jˆ6.1 Hz, Thr CgH3).
4.1.11. N-(Benzyloxycarbonyl) a-aminoisobutyryl-a-
aminoisobutyryl-O-(tert-butyl)-l-seryl-a-aminoisobu-
tyryl-a-aminoisobutyric acid methyl ester [Z±Aib±Aib±
Ser(tBu)± Aib±Aib±OMe] (5S). The title compound was
prepared from Z±Aib±Aib±OH (300 mg, 0.93 mmol) and
the N a-deprotected tripeptide 3S (320 mg, 0.93 mmol)
as described for the synthesis of 5TG by fragment
condensation (b), and obtained as a white solid after
puri®cation by MPLC (98: 2 CH2Cl2/MeOH). Yield
120 mg (20%); Rf (E6) 0.76; Rf (E8) 0.86; mp 183±
1848C; (Found: C, 58.97; H, 8.14; N, 10.56%.
C32H51N5O9 requires C, 59.15; H, 7.91; N, 10.78%);
[a]Dˆ119.5 (c ˆ0.7, MeOH); dH (400 MHz, CDCl3):
7.36 (6H, m, Ph CH, Ser NH), 7.30, 7.10, 6.67, 5.18
(4H, 4s, 4 Aib NH), 5.12 (2H, q, CH2Ph), 4.15 (1H, m,
Ser CaH), 3.81 (1H, m, Ser CbH), 3.68 (3H, s, OMe
CH3), 3.64 (1H, m, Ser CbH0), 1.54±1.45 (24H, m, 8
Aib CbH3), 1.15 (9H, s, tBu CH3); MALDI±TOFMS:
[M1Na]1 found 673. C32H51N5O9 requires 649.8.
4.1.8. N-(Benzyloxycarbonyl)-a-aminoisobutyryl-O-(tert-
butyl)-l-threonyl-a-aminoisobutyryl-a-aminoisobutyric
acid methyl ester [Z±Aib±Thr(tBu)± Aib±Aib±OMe]
(4T). The N a-deprotected tripeptide 3T (0.39 g,
1.10 mmol) was reacted with (Z±Aib)2O according to the
procedure described above for 4TG, and the crude product
was puri®ed by MPLC (98: 2 CH2Cl2/MeOH) and obtained
as a white solid. Yield 0.57 g (89%); Rf (E6) 0.92; Rf (E8)
0.81; mp 135±1368C; (Found: C, 60.41; H, 8.04; N, 9.49%.
C29H46N4O8 requires C, 60.19; H, 8.01; N, 9.68%);
[a]Dˆ16.5 (c ˆ0.9, MeOH); dH (250 MHz, CDCl3): 7.39
(1H, s, Aib NH), 7.35 (5H, m, Ph CH), 7.19 (1H, d,
Jˆ4.8 Hz, Thr NH), 7.11 (1H, s, Aib NH), 5.25 (1H, s,
Aib NH), 5.09 (2H, q, CH2Ph), 4.28 (1H, m, Thr CbH),
4.04 (1H, m, Thr CaH), 3.70 (3H, s, OMe CH3), 1.59±
1.49 (18H, m, 6 Aib CbH3), 1.18 (9H, s, tBu CH3), 1.12
(3H, d, Jˆ6.0 Hz, Thr CgH3); MALDI±TOFMS:
[M1Na]1 found 601. C29H46N4O8 requires 578.7.
FT-IR absorption. FT-IR absorption spectra were recorded
with a Perkin±Elmer model 1720X spectrophotometer
¯ushed with nitrogen, equipped with a sample-shuttle
device, at 2 cm21 nominal resolution, averaging 16
scans for 10 and 1.0 mM sample concentrations, or 64
scans for 0.1 mM sample concentration. Solvent (base-
line) spectra were recorded under the same conditions.
Cells with path lengths of 0.1, 1.0, and 10 mm (with
4.1.9. N-(Benzyloxycarbonyl)-a-aminoisobutyryl-a-amino-

2442
M. Gobbo et al. / Tetrahedron 57 (2001) 2433±2443
9. Toniolo, C. CRC Crit. Rev. Biochem. 1980, 9, 1±44.
10. Rose, G. D.; Gierasch, L. M.; Smith, J. P. Adv. Protein Chem.
1985, 37, 1±109.
CaF2 windows) were used. Spectrograde deuterochloro-
form (99.8% D) was purchased from Merck. Elaboration
of the spectra with subtraction of the baseline and the
second derivative was carried out with the SpectraCalc
(Galactic) program.
11. Basu, G.; Kuki, A. Biopolymers 1993, 33, 995±1000.
12. Gobbo, M.; Crisma, M.; Toniolo, C.; Rocchi, R. In Peptides
 Â
1998; Bajusz, S., Hudecz, F., Eds.; Akademiai Kiado:
1
Nuclear magnetic resonance. The H NMR spectra were
Budapest, 1999; pp 266±267.
13. Crisma, M.; Gobbo, M.; Toniolo, C.; Rocchi, R. Carbohydr.
Res. 1999, 315, 334±338.
recorded at 298 K, with a Bruker model AM-400 spec-
trometer. Measurements were carried out in CDCl3
(99.96% D, CIL) and deuterated DMSO (99.9% d6,
Acros Organics) with tetramethylsilane as the internal
standard.
14. Jones, D. S.; Kenner, G. W.; Preston, J.; Sheppard, R. C.
J. Chem. Soc. 1965, 6227±6239.
15. Mc Gahren, W. J.; Goodman, M. Tetrahedron 1967, 23,
2017±2030.
16. Valle, G.; Formaggio, F.; Crisma, M.; Bonora, G. M.;
Toniolo, C.; Bavoso, A.; Benedetti, E.; Di Blasio, B.; Pavone,
V.; Pedone, C. J. Chem. Soc., Perkin Trans. 2 1986, 1371±
1376.
X-Ray diffraction. Single crystals of Z±Aib±Aib±
Thr(tBu)± Aib±Aib±OMe (5T) were obtained by slow
evaporation at room temperature from an ethanol solution.
Intensity data collection was performed using a Philips PW
1100 four-circle diffractometer. Graphite-monochromated
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Kates, S. A. Lett. Pept. Sci. 1994, 1, 57±67.
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Ê
Cu Ka radiation (lˆ1.54178 A) and u/2Q scan mode
were used. Cell parameters were obtained by least-squares
re®nements of the angular setting of 48 carefully centred
high angle re¯ections. The structure was solved by direct
methods (SHELXS 97 42program). Re®nement was carried
out by full-matrix block least-squares on F2, using all data,
19. Leplawy, M. T.; Jones, D. S.; Kenner, G. W.; Sheppard, R. C.
Tetrahedron 1960, 11, 39±51.
Â
20. Benedetti, E.; Pedone, C.; Toniolo, C.; Dudek, M.; Nemethy,
with all non-H atom anisotropic, by application of the
SHELXL 97
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181.
43
program. The positional parameters and
the anisotropic displacement parameters of the non
H-atoms were allowed to re®ne at alternate cycles.
H-atoms were calculated at idealized positions and during
the re®nement they were allowed to ride on their carrying
atom with Uiso set equal to 1.2 (or 1.5 for methyl groups)
times the Ueq of the parent atom. Crystallographic data have
been deposited at the Cambridge Crystallographic Data
Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ,
UK (fax: 144-1223-336-033; e-mail: deposit@ccdc.cam.
ac.uk) and copies can be obtained on request, free of charge,
by quoting the publication citation and the deposition
number 152568.
21. Schweizer, W. B.; Dunitz, J. D. Helv. Chim. Acta 1982, 65,
1547±1554.
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Acknowledgements
È
28. Gorbitz, C. H. Acta Crystallogr. 1989, B45, 390±395.
We thank Mr F. Cavaggion and Mr V. Moretto for skillful
technical assistance.
29. Dunitz, J. D.; Strickler, P. In Structural Chemistry and
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